Nitric Oxide Resets Kisspeptin-Excited Gnrh Neurons Via PIP2 Replenishment

Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment

Stephanie Constantina, Daniel Reynoldsa, Andrew Oha, Katherine Pizanoa, and Susan Wraya,1

aCellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke (NINDS), NIH, Bethesda, MD 20892

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 23, 2020 (received for review June 15, 2020) 2+ Fertility relies upon pulsatile release of gonadotropin-releasing rate (20). Under normal conditions, [Ca ]i oscillations are driven hormone (GnRH) that drives pulsatile luteinizing hormone secre- by bursts of action potentials (AP) (21, 22). Yet, AP are not 2+ tion. Kisspeptin (KP) neurons in the arcuate nucleus are at the necessary for the KP-evoked [Ca ]i response to occur, as it is center of the GnRH pulse generation and the steroid feedback driven by multiple effectors including transient receptor potential- control of GnRH secretion. However, KP evokes a long-lasting re- canonical channels (TRPC), voltage-gated calcium channels sponse in GnRH neurons that is hard to reconcile with periodic (VGCC), and inositol 1,4,5-trisphosphate receptors (InsP3R) (15, GnRH activity required to drive GnRH pulses. Using calcium imag- 16, 19, 23). Thus, the versatility of Kiss1r signaling pathway un- ing, we show that 1) the tetrodotoxin-insensitive calcium response derlies the functionality of KP projections along GnRH neuron evoked by KP relies upon the ongoing activity of canonical tran- processes (24), with KP locally applied on nerve terminals also 2+ sient receptor potential channels maintaining voltage-gated evoking a long-lasting increase in [Ca ]i (16). calcium channels in an activated state, 2) the duration of the While the long-lasting KP response is suitable to trigger the calcium response is determined by the rate of resynthesis of phos- preovulatory surge, it seems incompatible with the KNDy model phatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide termi- for tonic pulses. GnRH and LH pulses occur every ∼20 min in nates the calcium response by facilitating the resynthesis of PIP2 ovariectomized mice (25). Indeed, the KNDy model provides via the canonical pathway guanylyl cyclase/3′,5′-cyclic guanosine on-/off- signals for KNDy neurons, neurokinin B and dynorphin monophosphate/protein kinase G. In addition, our data indicate respectively, and an on-signal for GnRH neurons, KP. However, that exposure to nitric oxide after KP facilitates the calcium re- this model lacks an off-signal for GnRH neurons. KNDy neurons NEUROSCIENCE sponse to a subsequent KP application. This effect was replicated trigger GnRH/LH pulses via Kiss1r (26), but the “extinction” of using electrophysiology on GnRH neurons in acute brain slices. The KNDy neurons by dynorphin is not obligatory for the termina- interplay between KP and nitric oxide signaling provides a mech- tion of GnRH/LH pulses in rodents (27, 28), and dynorphin is anism for modulation of the refractory period of GnRH neurons lacking in human KP-neurokinin neurons (29). after KP exposure and places nitric oxide as an important compo- 2+ In fact, how the response in firing and [Ca ]i to KP relate to nent for tonic GnRH neuronal pulses. GnRH secretion is unknown. In acute coronal brain slices, the KP-evoked increase in firing at the cell body cannot be repeated fertility | GnRH | kisspeptin | pulse | nitric oxide (19, 30). In contrast, in acute sagittal brain slices, using fast scan cyclic voltammetry, the KP-evoked GnRH secretion from cells in onadotropin-releasing hormone (GnRH)-secreting neurons the preoptic area (POA) and fibers in the median eminence Gintegrate multiple physiological and environmental cues and (ME) can be triggered repeatedly (31). The difference in re- translate them into GnRH secretory patterns, to drive gonado- peatability at the cell body is puzzling. One explanation could be trophs, which in turn control the gonads. In both sexes, tonic GnRH pulses regulate gametogenesis and steroidogenesis via Significance luteinizing hormone (LH) and follicle-stimulating pulses. Fe- males require a GnRH surge to trigger LH surge and ovulation Hypothalamic neural networks control hormone secretion to [reviewed in (1, 2)]. However, disruption of the normal pulsatile maintain body function. For gonadotropin-releasing hormone GnRH secretion impairs fertility in both sexes (3). Although (GnRH) neurons, critical to fertility, release must be pulsatile. insufficient for optimal reproductive health (4), the direct action While it is clear that kisspeptin neurons trigger GnRH activity of kisspeptins (KP) on GnRH neurons, via the KP receptor and pulses, how GnRH neurons return to baseline activity be- – Kiss1r, is required for fertility (4 6). KP neurons play a critical fore the next stimulation is unknown. Here, we show 1) that regulatory role in the hypothalamic–pituitary–gonadal axis, in- the long-lasting response of GnRH neurons to kisspeptin depends cluding puberty onset (7–9), the preovulatory GnRH surge (5, 9, on the degradation of phosphatidylinositol 4,5-bisphosphate

10), and tonic GnRH pulses (6, 11). In rodents, two KP neuronal (PIP2) and its slow resynthesis keeping canonical transient re- subpopulations exist with distinct functions: the anteroventral ceptor potential channels opened and 2) a mechanism whereby

periventricular nucleus (AVPV) subpopulation, linked to pu- nitric oxide facilitates PIP2 resynthesis and GnRH neuron recov- berty onset [reviewed in (12)] and preovulatory surge [reviewed ery from kisspeptin activation. Defining the overlap in kisspeptin in (13)], and the arcuate nucleus (ARC) subpopulation, also and nitric oxide signaling pathways may provide an avenue for known as Kisspeptin-Neurokinin B-Dynorphin (KNDy) neurons, fertility treatment. linked to tonic pulses (reviewed in ref. 14). Exogenous KP at the GnRH cell body evokes a long-lasting Author contributions: S.C. designed research; S.C., D.R., A.O., and K.P. performed re- 2+ search; S.C. analyzed data; and S.C. and S.W. wrote the paper. increase in intracellular calcium levels ([Ca ]i) (15), often 2+ The authors declare no competing interest. leading to the summation of individual oscillations into [Ca ]i plateaus (15, 16). This observation is in agreement with an in- This article is a PNAS Direct Submission. crease in electrical activity where GnRH neurons in acute slices Published under the PNAS license. go from burst firing to tonic firing after KP application (17–19). 1To whom correspondence may be addressed. Email: [email protected]. One could argue that this prolonged response is an artifact of the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ exogenously applied KP. However, endogenously released KP by doi:10.1073/pnas.2012339118/-/DCSupplemental. AVPV stimulation also evokes a long-lasting increase in firing Published December 21, 2020.

PNAS 2021 Vol. 118 No. 1 e2012339118 https://doi.org/10.1073/pnas.2012339118 | 1of11 Downloaded by guest on September 24, 2021 Fig. 1. The tetrodotoxin-insensitive calcium response to KP-10 in GnRH neurons. (A) High-magnification images of GnRH neurons in explants identified by their morphology (Left), recorded with calcium imaging (Middle), and confirmed by immunocytochemistry (Right). Arrows indicate the same neurons throughout the experiment. (Scale bar, 20 μm in all panels.) (B) A heat map showing changes in [Ca2+] levels in 49 GnRH neurons recorded simultaneously. Neurons were perfused for 5 min in a control medium, and then TTX was added for the rest of the recording. After 5 min in TTX, the cells received a 2-min application of KP-10. Optical density (arbitrary units) was measured, with cells showing an increase in calcium levels changing toward yellow. (C) Examples of 2+ traces showing changes in [Ca ] levels (ΔF/F0) in five individual GnRH neurons (gray) and the average in black, evoked by 10 nM (Top) or 100 nM (Bottom)KP. The time of the arrow heads in C correspond to the time of arrow heads in D.(D) Average trace for changes in [Ca2+] levels (percent) evoked by 10 nM (open circles, n = 3 explants) and 100 nM (filled circles, n = 7 explants; reference trace for subsequent experiments). Each trace was normalized to its maximum to assess the recovery time at 50% (red horizontal line) for each dose (red vertical lines). The arrow heads represent the recovery period during which drugs were applied in subsequent experiments (i.e., once the response to KP had fully developed). (E) A bar graph depicting the average time (+SEM) at 50% recovery for explants used in D. The dotted line represents the value used as the reference (30.0 ± 1.4 min) without perturbations (KP 100 nM) for subsequent experiments. The average time at 50% recovery between 10 nM and 100 nM KP was not significantly different (nonparametric Mann–Whitney U test).

2of11 | PNAS Constantin et al. https://doi.org/10.1073/pnas.2012339118 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Downloaded by guest on September 24, 2021 a technical consequence of brain slicing or conventional patch integrates these KP inputs (32) and the calcium response is ac- clamp. Another explanation could be a dissociation between firing tion potential–independent (16). At 7 to 14 d in vitro, ∼90% of 2+ and [Ca ]i during the second KP application. In fact, the com- primary GnRH neurons from explants increase intracellular 2+ mon feature of the KP-evoked increase in [Ca ]i and GnRH calcium in response to KP and express Kiss1r messenger RNA secretion, at the GnRH neuron cell body and nerve terminal, is (15). The signaling pathway downstream Kiss1r identified in 7- that it is AP-independent (15, 16, 31). The current study uses div GnRH neurons is identical to the one identified in GnRH calcium imaging and electrophysiology to address the mechanisms neurons from adult brain slices (15). that allow 1) [Ca2+] in GnRH neurons to return to baseline after i Because, like the GnRH neuron distal process (16), pri- KP stimulation, and 2) GnRH neurons to respond to a second KP mary GnRH neurons from explants exhibit action potential– stimulation (i.e., repeatability) and shows nitric oxide as an important component for tonic GnRH neuronal pulses. insensitive calcium signaling under KP stimulation (15), they were used for calcium imaging experiment as a tool to specif- Results ically study the recovery from this specific action potential– GnRH/LH pulses are driven by kisspeptinergic inputs arising insensitive calcium signaling in a large number of GnRH from KNDy neurons (26). The GnRH neuron distal process neurons. NEUROSCIENCE

Fig. 2. Canonical transient receptor potential channels and subsequently voltage-gated calcium channels feed the TTX-insensitive calcium response to KP.(A–C) Examples of traces showing changes in [Ca2+] levels in five individual GnRH neurons (gray) and the average (color) under the indicated perturbation. The first vertical line marks treatment with TTX (arrow indicates presence for the remaining recording period), the second and third vertical lines indicate treatment with KP (gray, 2 min), and the fourth vertical line marks beginning of experimental perturbation (length of perturbation is shown on top). (Inset)Horizontalbar= 5min, 2+ vertical bar = 0.2 ΔF/F0. Below each graph is the average response for changes in [Ca ] levels (percent) evoked by KP 100 nM without perturbation (control from Fig. 1D, black circles) or with perturbation (colored circles). (A) 2-APB curtailed the calcium response to KP, indicating the role of canonical TRPC in the maintenance of the calcium response. (B, C) Transient removal of extracellular calcium or blockade of L-type (Nifedipine [Nif]) and N-type (ω-Conotoxin GVIA [Ctx GVIA]) VGCC dampened the calcium response to KP but only during the perturbation, indicating the TRPC were still in an activated state and that TRPC worked synergistically with VGCC to maintain the calcium response. (D) A bar graph showing the average time (+SEM) at 50% recovery for GnRH cells recorded in A–C. Note that blocking or activating protein phosphatases 1/2 with OKA or C2, respectively, or protein kinase C with BIM X was ineffective, and other TRPC blockers (SKF96365,La3+,or flufenamic acid [FFA]) were not as effective as 2-APB. All average times at 50% recovery with 100 nM KP + drug were compared to the average time with 100 nM KP alone (black bar). Statistical significances (P < 0.05) were indicated with asterisks (nonparametric Mann–Whitney U test), and P values were listed in Table 1.

Constantin et al. PNAS | 3of11 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment https://doi.org/10.1073/pnas.2012339118 Downloaded by guest on September 24, 2021 The Time Course of the KP-Evoked Calcium Response Is Constant. The channels (VGCC) contribute to the calcium influx during the ultimate goal is to understand the mechanisms that lead to long-lasting response, a mixture of nifedipine (Nif; 1 μM) and GnRH pulses. Since both KP-induced GnRH secretion (31) and Ctx GVIA (100 nM), blockers of L-type and N-type VGCC, calcium response to KP (15, 16, 19) are tetrodotoxin (TTX) in- respectively, was used. The mixture was applied for 10 min during sensitive, the paradigm chosen was as follows: 5 min in serum- the KP recovery period. Although it effectively depressed the free media (SFM) (cell culture medium) was followed by 5 min calcium plateau (Fig. 2 C and D, P = 0.0083), the calcium response in SFM+TTX (0.5 μM) to establish the baseline (Fig. 1 B and C). resumed after its removal. While this indicated the contribution of SFM+TTX+KP (10 or 100 nM) was applied for 2 min between VGCC to the calcium response, it also showed the ongoing acti- the 10th and 12th minutes and then washout in SFM+TTX for vated state of TRPC channels. 30 min. The calcium responses evoked by the two KP doses had the same time-course. The time to 50% recovery was not different Modulation of TRPC Channels by Nitric Oxide Decreases Recovery (Fig. 1 D and E, P = 0.3667). The dose of 100 nM was chosen for Time from KP Activation. The degradation of phosphatidylinosi- the rest of the experiments. Unless stated, pharmacological per- tol 4,5-bisphosphate (PIP2) is required for the activation of turbations were applied after the 17th minute with SFM+TTX to TRPC (37). Preventing the resynthesis of PIP2 increases the avoid interfering with the onset and development of the full duration of the KP-evoked TRPC current (37). To test whether KP response. the resynthesis of PIP2 also sets the time course of the KP response, phosphatidylinositol 4-kinase (PI4K) was blocked with Maintenance of Calcium Response to KP Is Dependent on TRPC wortmannin (10 μM). The time at 50% recovery was increased Channels and VGCC. The balance between protein kinase and (Fig. 3 A and D, P = 0.0278). When wortmannin was applied at a phosphatase activities is at the center of the well-characterized dose specific to phosphoinositide 3-kinase (PI3K, 10 nM), it had long-lasting phenomena, long-term potentiation, and long-term no effect (Fig. 3D, P = 0.7545). A dense fiber network containing depression (33, 34). In neurons, calcium influx activates the the neuronal nitric oxide synthase (nNOS) exists in the median calcium/calmodulin‐dependent protein kinase II (CaMKII). An eminence (41–43) and endothelial nitric oxide synthase (eNOS) autophosphorylation keeps the enzyme active. In vitro, both produces nitric oxide in the median eminence (44). Since nitric protein phosphatase 1 (PP1) and protein phosphatase 2A oxide modulates PIP2 and calcium responses to growth factors (PP2A) can dephosphorylate CaMKII (35) (i.e., reverse its cal- (45–47), and the time at 50% recovery was increased when PIP2 cium-independent activity). Since a physical association between resynthesis was blocked, we tested whether promoting PIP2 Kiss1r and PP2A was identified in a heterologous expression resynthesis with nitric oxide would facilitate the recovery from system (36), the role of PP2A on the KP-evoked calcium re- KP stimulation. A 30-min application of a nitric oxide–releasing sponse was tested with okadaic acid (OKA, 30 nM; inhibitor of compound, DEA/NO (100 μM, 12 min into solution at the time PP1 and PP2A). The application of OKA did not affect the time the perfusion starts), abruptly stopped the KP-evoked calcium course of the recovery (Fig. 2D, P = 0.3671). An activator of PP1 response (Fig. 3 B and D, P = 0.0041). To ensure the effect of and PP2, C2 ceramide (also known as N-acetoyl-D-erythro- nitric oxide was mediated by the stimulation of PIP2 resynthesis sphingosine; 20 μM), was also tested and had no effect upon the via PI4K, rather than inhibition of TRPC channels (48), DEA/ time course of the response to KP (Fig. 2D, P = 0.6061). NO was applied with wortmannin (10 μM) and failed to affect Multiple studies showed the ability of 2-aminoethoxydiphenyl the time course of the KP-evoked calcium response (Fig. 3 C and borate (2-APB) to interfere with the onset of the KP response in D, P = 0.4254). Finally, to test whether the KP-evoked calcium GnRH neurons (15, 16, 19). The fact that extracellular, but not response was transiently paused or terminated by nitric oxide, intracellular, 2-APB blocks KP-induced nonselective cation in- experiments were performed in which the application of DEA/ ward currents, identifies receptor-operated currents in the KP NO lasted for only 5 min. This 5-min application of nitric oxide response and TRPC channels as mediators rather than store- was sufficient to stop the KP-evoked calcium response (Fig. 4 A operated currents (37). To determine whether the maintenance and F, P = 0.0239). Notably, no rebound appeared after removal of the long-lasting calcium response to KP was dependent on of DEA/NO, indicating that the TRPC channels were in fact persistent activation of TRPC channels, 2-APB (75 μM) was deactivated. applied during the KP recovery period. Treatment with 2-APB Canonically, nitric oxide activates soluble guanylyl cyclase, decreased the time to reach 50% recovery (Fig. 2 A and D, P = which in turn produces 3′,5′-cyclic guanosine monophosphate 0.0083), consistent with activated TRPC channels feeding the (cGMP) (49). cGMP activates its downstream effector, PKG KP-induced calcium response. (50). The less classical alternative is nitric oxide–induced post- The Kiss1r downstream signaling pathway involves protein translational modifications such as S-nitrosylation and nitration kinase C (PKC) (15, 38). Some TRPC channels are modulated (51, 52). To define the signaling pathway activated with DEA/ by a diacylglycerol and PKC-dependent mechanism (39). Thus, NO, first we tested whether a membrane-permeable analog of the PKC inhibitor bisindolylmaleimide X (BIM X, 100 nM) was cGMP, 8-pCPT-cGMP (cGMP, 200 μM), could mimic the effect applied during the KP recovery period. BIM X did not change of DEA/NO. cGMP was applied for 5 or 10 min and in both the time to reach 50% recovery (Fig. 2D, P = 0.9333), indicating cases, it dampened the KP-evoked calcium response (Fig. 4 B, C, that PKC activity does not determine the duration of the KP and F, P = 0.0061 and P = 0.0083, respectively). Second, we response. tested whether the synthesis of cGMP mediated the effect of 2-APB is a reliable blocker for Ca2+ entry, less so for Ca2+ DEA/NO by blocking the guanylyl cyclase prior to its applica- release (40). To confirm the calcium response to KP required an tion. DEA/NO failed to inhibit the KP-evoked calcium response influx, regular SFM was switched to nominal calcium-free SFM in GnRH neurons pretreated for 17 min with a blocker of gua- (0 Ca2+) for 10 min. As predicted, the calcium response dropped nylyl cyclase, ODQ (30 μM) (Fig. 4 D and F, P = 0.7636). Both during perfusion with calcium-free SFM (Fig. 2 B and D, P = experiments point toward the classical nitric oxide pathway 0.0083). Notably, the calcium levels went back to control levels ending the KP-evoked calcium response. The common down- upon calcium reintroduction, indicating the persistence of the stream effector of PKG, RhoA/Rho kinase (ROCK), links PKG activated state of TRPC channels, with or without calcium flow. activity to PIP2 synthesis (47, 53). A 17-min preincubation of Cadmium, a broad-spectrum blocker of VGCC, and ω-con- GnRH neurons with Y-27632 (ROCK inhibitor, 10 μM) did not otoxin GVIA (Ctx GVIA) attenuate and antagonize the onset of prevent DEA/NO to terminate the KP-evoked calcium response KP response in GnRH neuron cell bodies (15) and nerve ter- (Fig. 4 E and F, P = 0.0204), suggesting a different effector than minals (16), respectively. To test whether voltage-gated calcium ROCK downstream PKG.

4of11 | PNAS Constantin et al. https://doi.org/10.1073/pnas.2012339118 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Downloaded by guest on September 24, 2021 NEUROSCIENCE

2+ Fig. 3. Resynthesis of PIP2 defines the duration of the TTX-insensitive calcium response to KP. (A–C) Examples of traces showing changes in [Ca ] levels in five individual GnRH neurons (gray) and the average (color) under the indicated perturbation. The first vertical line marks treatment with TTX (arrow, indicates presence for the remaining recording period), the second and third vertical lines indicate treatment with KP (gray, 2 min), and the fourth vertical line

marks beginning of experimental perturbation (length of perturbation is shown on top). (Inset) Horizontal bar = 5 min, vertical bar = 0.2 ΔF/F0. Below each graph is the average response for changes in [Ca2+] levels (percent) evoked by KP 100 nM without perturbation (control from Fig. 1D, black circles) or with

perturbation (colored circles). (A) Preventing the resynthesis of PIP2 by blocking PI4K/PI3K with Wortmannin [μM, micromolar range] prolonged the calcium response to KP. This effect did not occur when PI3K alone was inhibited (Wortmannin [nM, nanomolar range]). (B) Facilitating synthesis of PIP2 with nitric oxide (DEA/NO) shortened the calcium response to KP. (C) Blocking PI4K/PI3K with Wortmannin [μM] abolished the effect of DEA/NO. (D) The bar graph represents the average time (+SEM) at 50% recovery for explants used in A, B, and C. All average times at 50% recovery with 100 nM KP + drug were compared to the average time with 100 nM KP alone (black bar). Statistical significances (P < 0.05) were indicated with asterisks (nonparametric Mann– Whitney U test), and P values were listed in Table 1.

Nitric Oxide Exposure Facilitates a Second KP-Evoked Response in in between had a larger response to the second KP application, GnRH Cells. The increase in firing evoked by KP was rarely re- ∼25% increase (Fig. 5 A and B, P = 0.0039). This showed that peatable in GnRH neurons as measured in acute brain slices the mechanism we identified above (i.e., DEA/NO-induced PIP2 (54). Since the present studies demonstrate that nitric oxide fa- resynthesis and TRPC channel deactivation) facilitated the re- cilitates the time to 50% recovery to KP application in GnRH peatability of the KP calcium response. neurons, we applied KP twice, with and without DEA/NO be- The KP firing response also relies upon the activation of tween, to determine whether the repeatability of the KP-evoked TRPC channels and lack of repeatability in primary GnRH calcium response would be altered. KP was applied between the neurons in acute slices (19, 55). Thus, using this different model, 10 and 12th min and a second time between the 37 and 39th min we examined whether DEA/NO application would allow the KP (i.e., 25 min later). Notably, during the second application, KP firing response to repeat. We repeated this paradigm (KP twice, evoked a small decrease in the first KP-evoked calcium response (Fig. 5A). However, after its removal, the calcium response with or without DEA/NO in between) on GnRH cells in acute returned to levels similar to that recorded after the first KP brain slices, monitoring electrical activity with loose patch re- application (i.e., ∼5% increase of the first response added from cordings. To our surprise, the KP-evoked increase in firing was the second exposure to KP) (Fig. 5B). During the next paradigm, partially repeatable in cells which had returned to baseline ac- DEA/NO was applied between the 17th and 32nd min. As pre- tivity in the KP twice group without DEA/NO (Fig. 5C). How- viously described, DEA/NO dampens the first KP-evoked cal- ever, the application of DEA/NO allowed a larger response to cium response. In contrast to cells that receive KP twice, without occur with the second application of KP (Fig. 5 C and D, DEA/NO in between, cells that received KP twice with DEA/NO P = 0.0451).

Constantin et al. PNAS | 5of11 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment https://doi.org/10.1073/pnas.2012339118 Downloaded by guest on September 24, 2021 2+ Fig. 4. Nitric oxide facilitates PIP2 resynthesis via its canonical pathway. (A–E) Examples of traces showing changes in [Ca ] levels in five individual GnRH neurons (gray) and the average (color) under the indicated perturbation. The first vertical line marks treatment with TTX (arrow, indicates presence for the remaining recording period), the second and third vertical lines indicate treatment with KP (gray, 2 min), and the fourth vertical line marks beginning of

experimental perturbation (length of perturbation is shown on top). (Inset) Horizontal bar = 5 min, vertical bar = 0.2 ΔF/F0. Below each graph is the average response for changes in [Ca2+] levels (percent) evoked by KP 100 nM without perturbation (control from Fig. 1D, black circles) or with perturbation (colored circles). (A) Similar to 30-min DEA/NO application, a 5-min application of DEA/NO shortened the calcium response and it did not resume after the removal of DEA/NO, indicating the deactivation of TRPC channels. (B, C) The effect of DEA/NO was partially mimicked by a cell permeable analog of cGMP, 8-pGTP-cGMP. (D) The effect of DEA/NO was antagonized by pretreatment with ODQ, a blocker of guanylyl cyclase (GC), indicating the canonical nitric oxide signaling pathway via GC/cGMP/protein kinase G. (E) Blocking Rho kinase with Y27632 did not prevent the effect of DEA/NO. (F) The bar graph represents the average time (+SEM) at 50% recovery for explants used in A–C. All average times at 50% recovery with 100 nM KP + drug were compared to the average time with 100 nM KP alone (black bar). Statistical significances (P < 0.05) were indicated with asterisks (nonparametric Mann–Whitney U test), and P values were listed in Table 1.

Discussion However, a 2- to 5-min stimulation, sufficient to elicit a GnRH/ To date, the majority of studies examining the effect of KP on LH pulse in vivo (26), did not lead to Kiss1r internalization. In GnRH cells focus on the initiation of the KP response [reviewed our paradigm, the calcium response fully developed in GnRH in (56)]. The present study investigates how KP evokes a long- neurons within the 3 min following the removal of KP. Thus, this lasting response in GnRH neurons and how GnRH neurons can study focused on the signaling pathway downstream of the Kiss1r return to baseline to allow repetitive responses to KP, which is controlling return to baseline of GnRH neurons after KP the keystone for the KNDy model to work. We show that 1) the stimulation. ongoing activity of TRPC channels, subsequently activating L-/ Electrophysiological studies identified the phospholipase C N-type VGCC, maintains the calcium response, 2) the ongoing pathway downstream of the Kiss1r and implicated Ba2+-sensitive activity of the TRPC channels is determined by the kinetics of potassium channels, flufenamic acid (FFA)-sensitive TRP chan- PIP2 resynthesis, 3) nitric oxide accelerates PIP2 resynthesis and nels, 2-APB-sensitive TRP channels, and/or InsP3R in the KP re- terminates the KP response, via a protein kinase G-dependent sponse (19, 55). Studies using calcium imaging have shown that at mechanism, and 4) nitric oxide exposure between two subse- the cell body, the sarco/endoplasmic reticulum calcium transport quent exposures to KP enhances a second calcium and firing ATPase blocker, thapsigargin, is ineffective on the KP-evoked in- 2+ response. crease in [Ca ]i, suggesting that InsP3-dependent calcium stores Previous study using heterologous expression systems showed are not solely involved in the calcium response to KP (15). Both Kiss1r internalization over prolonged exposure to KP (57). 2-APB or FFA reduced the percentage of cells responding to KP

6of11 | PNAS Constantin et al. https://doi.org/10.1073/pnas.2012339118 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Downloaded by guest on September 24, 2021 Fig. 5. Exposure to nitric oxide facilitates a second response to KP. (A) Average traces for changes in [Ca2+] levels (percent) evoked by two exposures to KP 100 nM with (blue circles) or without (black circles) DEA/NO application in between KP application. TTX was added at the arrow and remained present for the whole recording period. (B) A bar graph showing the ratio between the two responses to KP (KP2/KP1) when not exposed to DEA/NO (black) or exposed to DEA/NO (blue) after KP1. The ratio of KP2/KP1 calcium responses was greater when DEA/NO was applied between KP application. Statistical significance (P < 0.05) is indicated with an asterisk (unpaired t test with Welch’s correction). (C) Two examples of electrophysiological recordings showing the first response and the second response to KP, when artificial cerebrospinal fluid (aCSF) only (Top trace) or DEA/NO (Bottom trace) was used in between KP applications. (bars: 2 min, 50 pA). (D) A bar graph showing the firing rate before and after KP1 and KP2, when aCSF only (black) or DEA/NO (blue) was used in between KP applications. The firing rates (aCSF,KP1, and washout [aCSF and NO/aCSF]) were similar between both paradigms (KP–KP [black] and KP–NO–KP [blue]), but the firing rate after KP2 was statistically greater

when DEA/NO was applied between KP applications. Statistical significance (P < 0.05) was indicated with an asterisk (nonparametric Mann–Whitney U test). NEUROSCIENCE

but neither totally abolished the response, suggesting that TRP TRPC (62) or VGCC (63) via S-nitrosylation. Similarly, S-nitro- channels are not the only components of the calcium response to sylation of β-arrestin could not account for accelerated internali- KP. Only 2-APB combine with TTX were effective, indicating the zation and a subsequent arrest of calcium response (64). In fact, facilitation of a plasma membrane component by TRPC channels the DEA/NO effect was partially mimicked by a membrane-per- (15). At the GnRH neuron nerve terminal, similar conclusions meant cGMP and blocked by a guanylyl cyclase inhibitor, sup- were reached (16). The only difference in the calcium response to porting the canonical NO–sGC–cGMP signaling pathway (65). KP between the GnRH neuron cell body and nerve terminal is the Although Rho-kinase is the link between PKG and PIP2 synthesis relative contribution of InsP3-dependent calcium stores (16). While (47), our data with 17-min Rho-kinase blocker pretreatment did the role of TRPC channels in the onset of the KP response, firing not show an effect and one would expect longer exposure to de- and calcium, is well documented (15, 16, 19, 37, 55), their ongoing crease PIP2 membrane content. Thus, further experiments are activity throughout the long-lasting response is an important new needed to identify the link between PKG and PIP2 synthesis. point to acknowledge while thinking about the reversal of the Notably, our data indicate that DEA/NO application between KP response. two KP applications enhances the second response to KP, both Consistent with previous data (16), our data show that VGCC calcium and firing. Surprisingly, in our hands, the KP effect on were activated and remained active throughout the KP response. GnRH neuron firing was repeatable in cells that had recovered, However, our experiments reveal that VGCC did not drive the at least partially, from the first KP application. This observation response, being downstream TRPC channels. Indeed, usually TRPC is in contrast with other studies (19, 30). One explanation might do not mediate a calcium response but work in synergy with VGCC be the method used. One study used gramicidin-perforated patch (58). Hence, for the GnRH neuron, a return to baseline activity clamp which is known to induce membrane lipid redistribution required deactivation of TRPC channels. On one hand, PKC is (66). The other study used whole-cell patch clamp which is known to facilitate the KP response (15, 37) and to regulate the known to alter PIP2, hence ionic channel function (67, 68), with activity of TRPC channels (39). On the other hand, PP2A associates current run-down being the apparent consequence of cytosol with Kiss1r in heterologous system (36), can be activated by calcium dialysis (69). However, the effect of RF9 on GnRH neurons is influx through TRPC channels (59), and is a well-known player in also mediated by Kiss1R and shown to be repeatable (70). Thus, neuronal long-lasting phenomena (33, 34). However, neither ma- the variable response of GnRH neurons to a second application nipulation impacted the duration of the calcium response. of KP remains unclear. However, the experiments in the present The KP-induced TRPC activation requires PIP2 depletion, study clearly indicate that nitric oxide allowed GnRH neurons to with TRPC4alpha being the sensor (37, 60). Hence, the logical generate a greater response to the second KP application. While reciprocal to deactivate TRPC would be the replenishment of many parameters for the DEA/NO application might need op- PIP2. Indeed, blocking PI4K (i.e., preventing the resynthesis of timization to achieve a full reset, it is obvious that nitric oxide 2+ PIP2) prolonged the calcium response, which is in agreement facilitated the second response to KP. One could argue [Ca ]i with an enhanced cationic current (37). In hippocampal cultures, remained higher in KP–KP than KP–NO–KP, even after KP2. 2+ 2+ nitric oxide increases membrane PIP2 (46, 61) and in GnRH Yet, the increase in [Ca ]i preKP2-postKp2 (Δ[Ca ]i) was neurons, nitric oxide terminated the KP-evoked calcium response. greater in KP–NO–KP than KP-KP (i.e., greater ratio KP2/KP1 When PI4K was blocked, DEA/NO had no effect, supporting the in KP–NO–KP than KP–KP). Return to baseline and changes in 2+ idea that it facilitates PIP2 resynthesis but does not directly inhibit [Ca ]i were judged more relevant because 1) neuropeptide

Constantin et al. PNAS | 7of11 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment https://doi.org/10.1073/pnas.2012339118 Downloaded by guest on September 24, 2021 Fig. 6. Proposed model for pulsatile GnRH/LH release controlled by KNDy neurons. KNDy neurons are an interconnected population of neurons in the arcuate nucleus that coexpress KP (purple), neurokinin B (green), and dynorphin (red). The division of KNDy neurons in the diagram is artificial and serves to depict ON- (Left) and OFF- (Right) signals. According to the KNDy model, GnRH neurons are activated by KP, ON-signal (round inputs), and Neurokinin B, and dynorphin participates in an autoregenerative mechanism driving bouts of activity within the KNDy population. Neurokinin B evokes neuronal excitation (ON-signal, round inputs). Subsequently, released dynorphin evokes neuronal inhibition (OFF-signal, flat inputs). This alternation of excitation and inhibition leads to episodic KP release onto GnRH neurons (blue). The contribution of each input into the episodic release of KP is depicted on the top right. The original KNDy model suggests that episodic KP release is sufficient to trigger pulsatile GnRH release. However, this model does not take into consideration that KP evokes a long-lasting calcium response in GnRH neurons that hampers the ability of GnRH neurons to respond to the next kisspeptinergic event. Here, we propose the addition of a third partner, neuronal nitric oxide synthase (NOS1)-expressing neurons (orange). In this model, NOS1-expressing neurons, activated by KNDy neurons, via KP or neurokinin B or other unknown coexpressed neuropeptides, release nitric oxide (OFF-signal, flat inputs) in the vicinity of GnRH neurons. Highly diffusible, nitric oxide provides a global reset of the signaling pathway components within GnRH neurons necessary for the next kisspeptinergic event. The contributionofeach input into the episodic release of GnRH is depicted on the bottom right, where NO release occurs sometime after kisspeptinergic stimulation (time break, //).

secretion responds to calcium dynamics (71) and 2) tonic se- nitrergic modulation of GnRH neuron cell body relates to the cretion is not sustainable as it leads to fatigue (72). preovulatory surge while nitrergic modulation of GnRH neuron The ability of nitric oxide to terminate the KP response is of distal process relates to GnRH pulsatility. In the preoptic area, particular importance. Nitric oxide, possibly one of the earliest the release of nitric oxide restrains GnRH neurons during the signaling molecules in evolution (73), is at the center of repro- estrogen-negative feedback, but increased nitric oxide produc- ductive neuroendocrinology (review in (50)). In females, nitric tion is a landmark for GnRH/LH surge during the estrogen- oxide regulates ovulation, likely through a nitrergic connection positive feedback (84). Kisspeptinergic stimulation of nNOS- between KP and GnRH neurons. Endothelial cells express expressing neurons in the preoptic area mimics their activation Kiss1r (74, 75) and median eminence fragments exhibit eNOS- during the estrogen-positive feedback (77). At the median emi- synthetized nitric oxide pulses whose frequency is compatible nence, nitric oxide stimulates GnRH release in males and with GnRH pulses (44), yet mice lacking eNOS are fertile (76). KP neurons contact nitric oxide-synthesizing neurons expressing Kiss1r (77), and nNOS-containing fibers contact GnRH neurons Table 1. Calcium imaging summary in the POA (78). Accordingly, female nNOS knockout are in- Drugs (n explants) Time at 50% recovery (min) P value fertile. However, males are also infertile (79), implying an ad- ditional interaction. A dense nNOS-containing fiber network KP 10 nM (n = 3) 27.0 ± 1.0 0.3667 exists in the median eminence (41–43). Some evidently belong to KP 100 nM (n = 7) 30.0 ± 1.4 — the hypothalamo-neurohypophysial system (80). However, while + OKA (n = 7) 32.3 ± 2.3 0.3671 the nNOS-containing fibers contacting GnRH cell bodies are +C2(n = 4) 30.5 ± 4.72 0.6061 glutamatergic, the nNOS-containing fibers contacting GnRH +BIMX(n = 3) 31.7 ± 4.8 0.9333 nerve terminals are GABAergic (81). These GABAergic nNOS + 2-APB (n = 3) 22.0 ± 1.2 0.0083* 2+ fibers likely come from the ARC since only this nucleus contains +0Ca (n = 3) 21.3 ± 2.3 0.0083* GABAergic nitrergic neurons, while other nuclei mostly contain + Nif + Ctx GVIA (n = 3) 19.7 ± 0.3 0.0083* glutamatergic nitrergic neurons (81). This population also stands + SKF96365 (n = 3) 26.7 ± 2.2 0.2333 = ± out by its resistance to NADPH-diaphorase staining, which is +La3+(n 3) 26.7 1.2 0.1500 = ± usually seen in nitric oxide positive cells (43, 82). The different + FFA (n 5) 33.6 1.2 0.1174 = ± sites of action for nitric oxide are supported by functional + Wort [nM] (n 4) 29.3 3.0 0.7545 = ± changes evoked by nitric oxide and its downstream cGMP/PKG + Wort [uM] (n 5) 38.4 2.7 0.0278* + NO 30 min (n = 7) 22.0 ± 1.3 0.0041* pathway: at the GnRH neuron cell body, nitric oxide inhibits = ± GnRH firing (78); at the GnRH neuron distal process, it facili- + Wort [uM] + NO 30 min (n 6) 34.2 4.4 0.4254 +NO5min(n = 5) 23.0 ± 3.0 0.0239* tates GnRH release (44, 83). = ± KP signaling pathways allow subcellular regions of GnRH + cGMP 5 min (n 4) 19.3 0.9 0.0061* + cGMP 10 min (n = 3) 20.7 ± 1.2 0.0083* neurons to have specific roles (2). The integration of KP inputs + ODQ + NO 30 min (n = 4) 33.0 ± 4.3 0.7636 at the GnRH neuron cell body is critical for the GnRH/LH + Y27632 + NO 30 min (n = 6) 21.7 ± 2.1 0.0204* surge, while the integration of KP inputs at the GnRH neuron distal process is critical for GnRH/LH pulses (32). Hence, *P < 0.05.

8of11 | PNAS Constantin et al. https://doi.org/10.1073/pnas.2012339118 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Downloaded by guest on September 24, 2021 Table 2. Drug list Chemical names Abbreviations Doses Targets Providers, catalog no.

Tetrodotoxin TTX 0.5 μMNa+ channel blocker Tocris, 1069 and Cayman, 14964 Kisspeptin-10 KP 10 or 100 Kiss1r agonist Tocris, 4243 and Phoenix nM Pharmaceuticals Inc., 048–56 Okadaic acid OKA 30 nM Protein phosphatase 1 and 2A inhibitor Cayman, 10011490

N-acetoyl-D-erythro-sphingosine C2 ceramide 20 μM Protein phosphatase A2 activator Cayman, 62510 Bisindolylmaleimide X BIM X 100 nM Protein kinase C inhibitor Cayman, 17511 2-Aminoethoxydi-phenylborane 2-APB 75 μM TRPC1, TRPC3, TRPC5, and TRPC6 blocker & IP3 Cayman, 64970 receptor blocker Nifedipine Nif 1 μM L-type voltage-gated calcium channel blocker Sigma, N7634 ω-conotoxin GVIA Ctx GVIA 100 nM N-type voltage-gated calcium channel blocker Sigma, C9915 SKF96365 SKF96365 30 μM TRPC6, TRPC7 blocker Tocris, 1147 Lanthanum La3+ 100 μM TRPC3, TRPC4/5, TRPC6, TRPC7 blocker Sigma, 449830 Flufenamic acid FFA 100 μM TRPC3, TRPC5, and TRPC7 inhibitor Sigma, F9005 Wortmannin Wort [nM] 100 nM PI3K inhibitor Cayman 10010591 Wortmannin Wort [μM] 10 μM PI4K inhibitor Diethylamine NONOate DEA/NO 100 μM NO donor Cayman 82100 8-pCPT-cGMP cGMP 200 μM GMP-dependent protein kinase activator Sigma, C5438 1H-[1,2,4]oxadiazolo[4,3-a] ODQ 30 μM NO-sensitive guanylyl cyclase inhibitor Cayman, 81410 quinoxalin-1-one Y27632 Y27632 10 μM ROCK-I and ROCK-II inhibitor MedChemExpress, HY-10583

females (44, 83) and estrogen enhances the amplitudes of nitric fluorescent protein (GnRH-GFP, MGI:6158458) male mice (2 to 4 mo old, oxide pulses, but not its frequency (44). To date, the stimulation gonad intact) were used for electrophysiological recordings (95) (SI Appen- of GnRH release by nitric oxide is explained by the retraction of dix). All procedures were approved by the National Institute of Neurological tanycyte end-feet (84). However, these nitric oxide–mediated Disorders and Stroke, Animal Care and Use Committee and performed in NEUROSCIENCE morphological changes occur only during proestrus to facilitate the accordance with NIH guidelines. GnRH/LH surge (85). Yet, the role of nitric oxide pulses at Calcium Imaging. Explants (14 to 18 d in culture) were used as previously the median eminence for GnRH/LH pulses was hypothesized (44). – described (15), and the phenotype of the recorded cells was confirmed with The present study focused upon the action potential insensitive immunocytochemistry for GnRH (Fig. 1A)(SI Appendix). signaling that normally occurs at the distal GnRH neuron process under kisspeptinergic inputs and its nitrergic modulation. Electrophysiology. Male mice were chosen to avoid the possible influence of To date, research has focused on how KP can evoke GnRH fluctuating circulating steroids. Electrophysiology was performed as previ- secretion looking at the onset of the response in GnRH neurons ously described (96) (SI Appendix). and how nitric oxide modulates GnRH secretion looking at its action upon firing rate at the GnRH cell body (50). However, an Statistical Analysis. anatomical relationship between KP fibers and NOS1 neurons in Calcium imaging analysis. Regions of interest were drawn around GnRH neu- the ARC is found in mouse, sheep, and nonhuman primate (77, rons. Changes in optical densities over time were detected in iVision and 86, 87). To our knowledge, no equivalent study exists in human analyzed in Matlab (Mathworks, Natick, MA). All files are processed with the but NOS1 is present in the infundibular nucleus (88) where minimum user inputs to avoid bias (SI Appendix). The graphs represent the ± human GnRH neurons receive the heaviest innervation (89). In mean SEM of independent recordings from at least three explants (N the present report, we highlight a common mechanism between represents the number of explants). For each trace, the time at which the average trace is midway from maximal response and baseline (time at 50% KP and nitric oxide, (i.e., degradation and resynthesis of PIP 2 recovery in minutes) was determined (Fig. 1 D and E). Experimental values respectively, independent of firing activity). While the calcium were compared to KP control (100 nM) values using a nonparametric response of GnRH neurons to KP requires depletion of PIP2 Mann–Whitney U test. All results are summarized in Table 1. (37), a subsequent response requires its resynthesis. The rate of To test the repeatability of the KP response, KP was applied on explants the PIP2 replenishment sets the refractory period for the effi- twice (i.e., after a 25-min washout period) with and without DEA/NO (100 ciency of the next response to KP. Nitric oxide facilitates the next μM) in between (SI Appendix). The amplitude of the responses was deter- response to KP, by facilitating PIP2 resynthesis, subsequently mined by subtracting the baseline value to the response value, and the deactivating TRPC channels and restoring their availability. For amplitude of the second application was normalized to the amplitude of the appropriate timing of KP/nitric oxide interplay, nitric oxide first application. Unpaired nonparametric Mann–Whitney U test was used to might be synthetized by NOS1 neurons in response to a Kiss1 compare the second KP response. neuron signal, the location of these NOS1 neurons remains to Electrophysiology analysis. Analysis was performed with Clampfit 10 over 5-min determined. However, given its unique rapid and unrestrained windows before and after KP. KP was applied twice, with or without DEA/ μ diffusibility, nitric oxide might provide the break for the calcium NO (100 M) in between (SI Appendix). The responses to the second KP response in a pool of GnRH neurons simultaneously and restore application between cells exposed or not to DEA/NO were compared using an unpaired t test with Welch’s correction. their ability to respond to the next kisspeptinergic stimulation (Fig. 6). Thus, the PIP2 signaling overlap between KP and nitric Drugs. The drugs (name, abbreviation, doses, target, and providers) are listed oxide may be an essential component for GnRH neuron pulses in Table 2. and provide the response to the long-sought-after question (90–93) (i.e., synchronization within the GnRH neuronal population). Data Availability. All study data are included in the article and SI Appendix.

Materials and Methods ACKNOWLEDGMENTS. This work was supported by the Intramural Research Animals. Embryos collected from timed-pregnant NIH Swiss mice were used Program of the NIH, National Institute of Neurological Disorders and Stroke to generate primary explant cultures (94) (SI Appendix). Adult GnRH-green (Grant ZIA-NS-002824).

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